MEF derivation

Primary MEFs were derived from F1 FVBxC57BL/6J embryos at 13.5 days post coitum (dpc) (Behringer et al., 2014). MEF culture medium consisted of 89% DMEM (Invitrogen), 10% fetal bovien serum (FBS; Hyclone), 1% penicillin and streptomycin (100 U/100 µg). For feeder preparation, γ-irradiated MEFs (20 Gy) were plated into six-well plates precoated with 0.1% gelatin at density 2×10⁴/cm². Feeder was used for mESC culture on the next day after cell plating. The standard NIH 3T3 protocol was used for establishing immortal MEF cell lines.